Hematopoiesis in the Bone Marrow FADD Deficiency Impairs Early

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FADD Deficiency Impairs Early Hematopoiesis in the Bone Marrow Stephen Rosenberg, Haibing Zhang and Jianke Zhang This information is current as J Immunol 2011; 186:203-213; Prepublished online 29 of September 27, 2021. November 2010; doi: 10.4049/jimmunol.1000648 http://www.jimmunol.org/content/186/1/203 Downloaded from

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FADD Deﬁciency Impairs Early Hematopoiesis in the Bone Marrow

Stephen Rosenberg, Haibing Zhang, and Jianke Zhang

Signal transduction mediated by Fas-associated death domain protein (FADD) represents a paradigm of coregulation of apoptosis and cellular proliferation. During apoptotic signaling induced by death receptors including Fas, FADD is required for the recruitment and activation of caspase 8. In addition, a death receptor-independent function of FADD is essential for embryogenesis. In previous studies, FADD deficiency in embryonic stem cells resulted in a complete lack of B cells and dramatically reduced T cell numbers, as shown by Rag12/2 blastocyst complementation assays. However, T-specific FADD-deficient mice contained normal numbers of thymocytes and slightly reduced peripheral T cell numbers, whereas B cell-specific deletion of FADD led to increased peripheral B cell numbers. It remains undetermined what impact an FADD deficiency has on hematopoietic stem cells and

progenitors. The current study analyzed the effect of simultaneous deletion of FADD in multiple cell types, including bone Downloaded from marrow cells, by using the IFN-inducible Mx1-cre transgene. The resulting FADD mutant mice did not develop lymphoprolifera- tion diseases, unlike Fas-deficient mice. Instead, a time-dependent depletion of peripheral FADD-deficient lymphocytes was observed. In the bone marrow, a lack of FADD led to a dramatic decrease in the hematopoietic stem cells and progenitor- enriched population. Furthermore, FADD-deficient bone marrow cells were defective in their ability to generate lymphoid, myeloid, and erythroid cells. Thus, the results revealed a temporal requirement for FADD. Although dispensable during lym-

phopoiesis post lineage commitment, FADD plays a critical role in early hematopoietic stages in the bone marrow. The Journal of http://www.jimmunol.org/ Immunology, 2011, 186: 203–213.

poptosis plays a critical role in mammalian development downstream caspases and other cellular proteins, leading to cell and homeostasis (1, 2). The intrinsic apoptotic signaling death. A is mediated by the mitochondrion, involving cytochrome The importance of apoptosis in development is exemplified by C and the Bcl-2 family proteins (3). The extrinsic apoptotic the embryonic defects caused by a lack of Bcl-x or Mcl-1, members pathways are initiated by ligation of death receptors (DRs) by of the Bcl-2 family that mediate the intrinsic pathway (12, 13). either their cognate ligands or cross-linking Abs (4, 5). DRs, in- Deficiencies in proapoptotic Bcl-2 family members such as Bax cluding TNF-R1, Fas/Apo-1, and TRAIL-Rs (DR4 and DR5), and Bak resulted in interdigital webbing due to insufficient death by guest on September 27, 2021 activate a caspase cascade through the adaptor protein Fas- of superfluous cells (14, 15). Deletion of another proapoptotic Bcl- associated death domain protein (FADD), which recruits procas- 2 family member, Bim, leads to autoimmune diseases caused by pase 8 to form the death-inducing signaling complex (DISC) (6– impaired death of autoreactive lymphocytes (16). The extrinsic 11). The assembly of the DISC promotes the activation of caspase pathways are essential for maintaining homeostasis in the immune 8 by self-processing, and the resulting active caspase 8 cleaves system. In particular, a systemic loss of Fas leads to the development of an age-dependent lymphoproliferative (lpr) and autoimmune disease (17). Although expressed in a wide range of tissues, DRs do not appear Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas to play an overt role in mouse development (18–22). Interestingly, Jefferson University, Philadelphia, PA 19107 mice deficient in FADD or caspase 8 die in utero by day 9.5–10.5 Received for publication February 24, 2010. Accepted for publication October 26, 2010. of gestation (23–25). Conditional deletion of FADD or caspase 8 This work was supported in part by National Institutes of Health Grants C0A95454, following lineage commitment in double-negative thymocytes or AI083915, and AI076788, a W. W. Smith Charitable Trust grant, a Thomas Jefferson pro-B cells resulted in no significant defects in the maturation of T University Enhancement grant, and a Concern Foundation grant to J.Z. S.R. was or B cells within primary lymphoid organs (26–30). When tested supported by a National Research Service Award Training grant (T32-AI07492). in vitro, FADD2/2 or caspase 82/2 lymphocytes are defective in Address correspondence and reprint requests to Dr. Jianke Zhang, Department of 2/2 Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson Univer- death receptor-induced apoptosis. Additionally, FADD and 2/2 sity, 233 South 10th Street, Room 731 BLSB, Philadelphia, PA 19107. E-mail address: caspase 8 T cells failed to expand efficiently upon stimulation [email protected] through the TCR, whereas FADD2/2 and caspase 82/2 B cells are The online version of this article contains supplemental material. impaired in TLR-induced proliferation. The effect of FADD de- Abbreviations used in this paper: BFU-E, erythroid burst-forming unit; CFU-GEMM, ficiency on the development and function of myeloid cells and granulocytic-erythrocytic-megakaryocytic-macrophagic CFU; CFU-GM, granuloma- hematopoietic progenitors has not been determined. crophagic CFU; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; DC, dendritic cell; DISC, death-inducing signaling complex; DR, death The process of hematopoiesis commences with hematopoietic receptor; EPO, erythropoietin; ES, embryonic stem; FADD, Fas-associated death stem cells (HSCs) (31). HSCs are located in the endosteal niche domain protein; GMP, granulocytic-macrophage progenitor; HSC, hematopoietic 2 + 2 + 2 stem cell; LCCM, L-cell conditioned media; LK, Lin c-Kit Sca-1 ; lpr, lymphopro- within the bone marrow and are characterized as Lin Sca-1 c- 2 + liferative; LSK, Lin Sca-1+c-Kit+; MEP, megakaryocytic-erythroid progenitor; MPP, Kit (32). HSCs proceed to differentiate into multipotent pro- multipotent progenitor; Mut, mutant; poly(I:C), polyinosinic-polycytidylic acid; rm, genitor (MPP) cells, which subsequently develop into either recombinant mouse; SCF, stem cell factor; WT, wild-type. common lymphoid progenitors (CLPs) or common myeloid pro- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 genitors (CMPs) (33, 34). The differentiation of MPPs indicates www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000648 204 FADD FUNCTION IN EARLY HEMATOPOIESIS the branching point at which developing hematopoietic cells on a MoFlo cell sorter (DakoCytomation, Carpinteria, CA). To analyze commit to the lymphoid or myeloid lineage. CLPs develop into lymphocyte populations, cells were washed once with staining buffer precursors for the T, B, and NK cell populations, whereas CMPs and labeled with the following lineage-specific fluorochrome-conjugated Abs: CD4-PE, CD8-TriColor, B220-TriColor, streptavidin-R670 (Caltag- further differentiate into the granulocytic-macrophage progenitors Invitrogen), CD5-PE (eBioscience), CD3-PE, CD19-Bio, c-Kit–PE, CD25- (GMPs) and megakaryocytic-erythroid progenitors (MEPs) (33, PE, IgD-PE (BD Pharmingen), IgM-FITC (Jackson ImmunoResearch 35). GMPs differentiate into macrophages and granulocytes, Laboratories), IgM-PE, IgD-Bio (Southern Biotechnology Associates, whereas MEPs are capable of differentiation into megakaryocytes Birmingham, AL). Cells were analyzed using a Coulter Epics XL cy- tometer (Beckman Coulter, Fullerton, CA). FlowJo (Tree Star, Ashland, and erythrocytes (36, 37). Both CLPs and CMPs lead to dendritic OR) was used for the generation of histograms and dot plots. For sorting, cell (DC) differentiation; therefore, DCs can be of either myeloid bone marrow cells were resuspended in sorting medium (1:1 PBS/RPMI or lymphoid origin (34, 38, 39). 1640). GFP2 bone marrow cells were isolated using a MoFlo high-speed Apoptosis, proliferation, and differentiation of HSCs and pro- cell sorter (DakoCytomation) in the Flow Cytometry Facility at Thomas genitors are tightly regulated. For example, a severe impairment of Jefferson University. hematopoiesis occurs following the loss of antiapoptotic functions L-cell conditioned media mediated by proteins of the intrinsic pathway, including Bcl-x and We used a protocol as described previously (49). L929 cells were purchased Mcl-1 (12, 40–42). The function of proteins of the extrinsic ap- from American Type Culture Collection (Manassas, VA) and cultured optotic pathways has also been investigated. Whereas there has in DMEM (Mediatech, Washington, DC) supplemented with 10% FBS been no HSC defect reported in mice lacking individual DRs, (Mediatech), 100 U/ml penicillin, and 100 mg/ml streptomycin (Medi- inducible deletion of caspase 8 resulted in greatly diminished atech). Confluent cells were detached with 3 ml solution containing 0.25% trypsin and 2.21 mM EDTA, washed with DMEM, and counted. Cells were in vitro differentiation of hematopoietic progenitor cells (43). In resuspended at a density of 2.4 3 105 in 58 ml DMEM, plated in a 175 cm3 Downloaded from a previous study, the effect of dominant-negative mutants of tissue culture flask, and incubated at 37˚C with 10% CO2 for 7 d. On day 7, FADD and caspase 8 on fetal liver progenitor cells was analyzed (44). However, the impact of simultaneous deletion of FADD in early hematopoietic lineages has not been formally tested. In this study, we used a Cre recombinase under the control of the Mx1

promoter to induce the deletion of FADD in multiple cell types http://www.jimmunol.org/ including bone marrow cells. These results help establish that FADD is essential at early stages of hematopoiesis, as its deletion in bone marrow cells impaired the peripheral lymphoid, myeloid, and erythroid lineages.

Materials and Methods Mice and primary cell isolation

FADD:GFP mice have been described (26, 27) and were crossed to mice by guest on September 27, 2021 bearing the Mx1-cre transgene purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in germ-free rooms in Thomas Jefferson University research animal facilities (Philadelphia, PA). The procedures were approved by the Institutional Animal Care and Use Committee. Excision of the FADD:GFP transgene in mice bearing the Mx1-cre transgene was induced via injection of the dsRNA polyinosinic- polycytidylic acid [poly(I:C); Sigma-Aldrich, St. Louis, MO; InvivoGen, San Diego, CA]. Mice were injected three times with 400 mg poly(I:C) in 200 mlH2O i.p. every other day as described previously (42, 45–47). At the indicated times after the last injection, cells were isolated from the thymus, spleen, lymph nodes, femurs, and tibias. The peritoneal cavity was lavaged with PBS. RBCs were lysed hypotonically with ACK lysis buffer. Cells were washed two to three times with PBS and counted using a hemocytometer.

Flow cytometry and cell sorting To detect HSCs, bone marrow cells were washed with staining buffer (3% BSA, 1 mM EDTA, 0.05% NaN3 in PBS) and stained with the following FIGURE 1. Inducible deletion of FADD:GFP using the Mx1-cre system. Abs; Sca-1–PeCy5 (eBioscience, San Diego, CA), c-Kit–PE, and an A, Single-cell suspensions prepared from the indicated organs were subject Allophycocyanin-Lineage Cocktail (BD Pharmingen, San Diego, CA), and to flow cytometric analysis, and presence and absence of GFP was illus- four-color flow cytometric analysis was performed on an FACSCalibur 2 2 trated in histograms. FADD+/ mice were used as GFP controls, and (BD Biosciences, San Jose, CA) due to FADD:GFP expression. Deletion 2/2 flox was assessed by determining the GFP2 population present in the c-Kit+ FADD FADD:GFP mice, uniformly expressing FADD:GFP in cells 2 + + 2 of various hematopoietic organs as indicated by a discrete GFP-positive Lin or Sca-1 c-Kit Lin population as previously described (42, 48). For 2 lymphoid progenitor analyses, bone marrow cells were stained with peak, were used as GFP+ controls. FADD+/ FADD:GFPflox Mx1-cre 2 2 allophycocyanin-conjugated lineage mixture, c-Kit–PE-Cy7 (BD Phar- control mice expressing an endogenous WT allele of FADD and FADD / mingen), Sca-1–PE-Cy5, and IL-7Ra-PE (eBioscience) (five-color analy- FADD:GFPflox Mx1-cre mutant mice were injected with poly(I:C). De- ses including GFP). To analyze myeloid and erythroid progenitor pop- letion of FADD:GFP in various organs was indicated by the presence of ulations, bone marrow cells were stained with Allophycocyanin-Lineage GFP2 cells 2 wk after the final injection. Numbers indicate percentages of Cocktail (BD Pharmingen), c-Kit–PE-Cy7, CD16/CD32/FcgRII/III-PE, GFP2 cells. The data show a representative of five control and mutant biotinylated Sca-1 (eBioscience), streptavidin-PE–Texas Red (Caltag- mice. The relative deletion efficiencies across mice (average 6 SD) was Invitrogen, Carlsbad, CA), and CD34–PE-Cy5 (Biolegend, San Diego, 2 CA) (six-color analysis including GFP). In some experiments, purified described the results. B, Western blotting showed that sorted GFP bone anti-CD34 (BD Pharmingen)/anti-rat–IgG-Texas Red (Jackson Immuno- marrow cells and spleen cells contained undetectable FADD:GFP protein. Research Laboratories, West Grove, PA) and Sca-1–PE-Cy5 (eBioscience) Protein transfer was confirmed by staining the nitrocellulose membrane were used. These six-color analyses of progenitors were performed with Ponceau S (Sigma-Aldrich). The Journal of Immunology 205

DMEMfromtheprevious7dwasfilteredthrough0.45-mm filters 2 d, and on day 8, cells in plates were photographed with a Nikon digital (Corning, Corning, NY) and stored at 220˚C as week 1 L-cell conditioned still camera (Model #DXM1200, Nikon) on a Nikon Eclipse TS100 media (LCCM). Fresh DMEM (58 ml) was added to the flasks and in- inverted microscope (Nikon). DC yield was assessed on day 8 by cubated for an additional 7 d. On day 14, the DMEM was filtered and counting cells by trypan blue exclusion and staining with anti–CD11b-PE stored at 220˚C as week 2 LCCM. This media was added to DMEM to and anti–CD11c-FITC (BD Biosciences). make bone marrow macrophage media (see below). In vitro CFU assays Preparation of bone marrow-derived macrophages This assay was performed as described (54, 55). GFP2 bone marrow cells The protocol was based on those described previously (22, 50). Bone were isolated by sorting and washed two times with IMDM (Life Tech- marrow cells were resuspended (106/ml) in DMEM (Mediatech) con- nologies, Rockville, MD) containing 2% FBS. Cells were diluted to 2 3 taining 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 105/ml in IMDM-2% FBS. For each dish, 2.5 3 104 cells were mixed with 30% LCCM (15% week 1 LCCM, 15% week 2 LCCM). Cells were 1 ml methylcellulose medium containing 50 ng/ml recombinant murine cultured for 5 d at 37˚C with 5% CO2 in 10-cm plates. Fresh media was stem cell factor (rmSCF), 10 ng/ml rmIL-3, 10 ng/ml rhIL-6, and 3 U/ml added on day 5, the media was replaced on day 6 to remove nonadherent erythropoietin (EPO; M3434, StemCell Technologies, Vancouver, British cells, and on day 7, plates were photographed with a Nikon digital still Columbia, Canada) and plated in 35-mm cell culture dishes using a 3 ml camera (Model #DXM1200, Nikon, Melville, NY) on a Nikon Eclipse syringe and a 16-gauge blunt-end needle. Assay plates were cultured at TS100 inverted microscope (Nikon). Adherent cells were removed by 37˚C with 5% CO2 for 10–14 d. Plates were then scored for erythroid incubating with 10 mM EDTA in PBS as described (51) and counted burst-forming units (BFU-E), granulocytic-erythrocytic-megakaryocytic- using a hemocytometer. macrophagic CFUs (CFU-GEMM), and granulomacrophagic CFUs (CFU- GM) by microscopic analysis based upon morphological appearance using Preparation of bone marrow-derived DCs a gridded scoring dish (StemCell Technologies).

The protocol was modified from those published previously (52, 53). Bone Splenic CFU assays Downloaded from marrow cells were diluted to 106/ml in RPMI 1640 (Mediatech) supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 10 The protocol was based on those described (43, 54). Bone marrow cells ng/ml rGM-CSF (R&D Systems, Minneapolis, MN), and 10 ng/ml rIL-4 were counted, and 1 3 105 cells were injected retro-orbitally in 200 ml 137 (R&D Systems) plated for 6 d with 5% CO2. On day 6, the top 80% of the PBS into irradiated recipient C57BL/6 (B6) mice (8.5 Gy, [ Cs] source). media was discarded and the bottom 20% used to wash the semiadherent Mice were treated with Sulfatrim (Mutual Pharmaceutical, Philadelphia, cells from the bottom of the plate, without disturbing the macrophage PA) over the course of the experiment. Mice were sacrificed 8 d post- monolayer. The resulting cell suspension was added to fresh RPMI 1640 transplantation. Spleens were photographed and weighed, then fixed in supplemented with 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomy- Bouin’s fixative (75 ml picric acid, 25 ml formalin, 5 ml glacial acetic http://www.jimmunol.org/ cin, 10 ng/ml rGM-CSF, and 10 ng/ml rIL-4 and plated for an additional acid) (43, 56). Colonies were counted following fixation. by guest on September 27, 2021

FIGURE 2. FADD deletion led to reduced hematopoietic pools. A, Total cell numbers of the indicated organs from pairs of WT and FADD mutant (Mut) were determined at 4 d after the last dose of poly(I:C) injection. Horizontal bars indicate the mean values. B, Mice of the indicated genotypes were injected with poly(I:C), and 2 wk after the ﬁnal injection, ﬂow cytometric analysis of splenic and lymph node cells were performed. Deletion of FADD:GFP in CD3+ T cells and B220+ B cells was indicated by the production of the GFP2 population. Numbers indicate the percentages of lymphocyte lineages and the percentages of the GFP2 population in each lineage in the indicated organs. 206 FADD FUNCTION IN EARLY HEMATOPOIESIS

Mouse hematocrit assays crossed into FADD2/2 FADD:GFPflox mice. Systemic administration of poly(I:C) was carried out in the resulting FADD2/2 FADD: Following administration of poly(I:C), mice were retro-orbitally bled using flox +/2 flox heparinized microhematocrit capillary tubes (22-362-566, Fisher Scientific, GFP Mx1-cre mutant mice and FADD FADD:GFP Mx1- Waltham, MA). Capillary tubes were sealed with Critoseal (McCormick/ cre control mice, which contain one allele of endogenous FADD. Fisher Scientific), centrifuged for 5 min in a Readacrit centrifuge (Clay The Mx1 promoter can be activated by stimulation with type I IFNs, Adams, Analytical Scientific, San Antonio, TX), and RBC percentage was or endogenous IFN can be induced by injection of the double- measured by dividing RBC volume from total volume (57). stranded viral RNA mimic poly(I:C) (45). At 14 d postinduction 2 2 Rag-1 / adoptive transfer experiments of deletion, cells from the bone marrow, thymus, spleen, and lymph GFP2 bone marrow cells (1 3 106) in 200 ml PBS were injected retro- nodes were isolated and analyzed by flow cytometry. FADD:GFP orbitally into sublethally irradiated Rag-12/2 recipient mice (4 Gy, [137Cs] is ubiquitously expressed in bone marrow cells, thymocytes, and source). Mice were kept on Sulfatrim (Mutual Pharmaceutical) and sac- peripheral cells as detected by flow cytometric analyses (Fig. 1). As rificed 8 wk postinjection. Organs were harvested and analyzed by flow reported in previous studies using these mice (26, 27), deletion of cytometry. FADD:GFP was readily detectable by flow cytometry, as indicated by the presence of a GFP2 population in control and mutant mice Results injected with poly(I:C), as well as by Western blotting (Fig 1A,1B). Inducible deletion of FADD The relative deletion efficiencies across mice (average 6 SD) range We previously described the FADD2/2 FADD:GFPflox mice in as follows: bone marrow, control 79.5–93.1% (87.1 6 5.9%) and which a floxed FADD:GFP fusion transgene corrected the de- mutant 67.6–92.1% (82.3 6 10.4%); thymus, control 60.8–98.5% velopmental defect caused by deletion of the endogenous FADD (84.78 6 17.2%) and mutant 83.1–94.3% (89.8 6 4.8%); spleen, Downloaded from alleles (23, 26). Lineage-specific deletion of FADD:GFPflox in pro/ control 54.3–62.8% (58.1 6 4.1%) and mutant 40.7–61.7% (55.8 6 pre-T and -B cells was achieved using the Lck-cre and CD19-cre 10.1%); and lymph nodes, control 40.8–57.4 (51.9 6 5.9%) and transgenes, respectively (26, 27). To analyze the effect of simulta- mutant 40.0–56.0% (47.8 6 6.8%) (n =5). neous deletion of FADD in multiple cells types in adult mice, in- Total cell numbers in various hematopoietic organs were de- cluding bone marrow progenitor cells, the Mx1-cre transgene was termined. When compared with FADD+/2FADD:GFPflox Mx1-cre http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Time-dependent depletion of FADD2/2 cells. Three months after the final injection of poly(I:C), cells of the indicated organs were subject to flow cytometric analyses. Percentages of GFP2 FADD2/2 mutant cells in the total organs (A) and in the peripheral lymphocyte populations (B, C)in FADD2/2 FADD:GFPflox Mx1-cre mutant mice were dramatically decreased when compared with the percentages of GFP2 FADD+/2 cells in the FADD+/2FADD:GFPflox Mx1-cre control mice. The data shown are representative of five control and mutant mice. The Journal of Immunology 207 control mice, a significant decrease in the total cellularity in the nodes (Fig. 3B) than control mice at 2 wk postinduction of de- bone marrow and thymus was observed in FADD2/2 FADD: letion (Fig. 2B). In contrast, FADD mutant mice contained greatly GFPflox Mx1-cre mutant mice (Fig. 2A). FADD deficiency also reduced numbers of FADD2/2 T cells (GFP2, 7.5%) and B cells resulted in somewhat lower numbers of splenocytes. The per- (GFP2, 15%) (Fig. 3B, Supplemental Fig. 1). These results in- centages of double-negative, double-positive, and single-positive dicate that FADD is required for the maintenance of various populations of thymocytes in FADD2/2 mutants were similar to hematopoietic cells in the bone marrow, thymus, and periphery. those in control mice (data not shown). Analysis of splenocytes 2 2 In vitro myeloid and lymphoid cell derivation from FADD / and lymph node cells showed a mild increase in B220+ B cells in bone marrow cells FADD2/2 FADD:GFPflox Mx1-cre mutant mice as compared with 2/2 FADD+/2 FADD:GFPflox Mx1-cre control mice (Fig. 2B). Con- Depletion of FADD cells in the bone marrow and periphery versely, fewer CD3+ T cells were found in mutant mice than in may be due to defects in hematopoietic progenitor cells. To test controls. this hypothesis, in vitro differentiation assays were performed. 2/2 Bone marrow macrophage progenitor cells can be induced to Time-dependent depletion of FADD cells differentiate into mature macrophages in vitro by culturing them Recent studies have shown that conditional deletion of Fas using in the presence of M-CSF (49, 59). GFP2 bone marrow cells were CD11c-cre led to an lpr-like phenotype, suggesting a role for Fas- sorted from FADD+/2 FADD:GFPflox Mx1-cre control and induced apoptosis in nonlymphoid cells (58). Therefore, FADD FADD2/2 FADD:GFPflox Mx1-cre mutant mice and cultured in mutant mice were aged for 3 additional mo following poly(I:C) conditioned medium containing M-CSF as described (22). As seen +/2 injections, yet they did not appear to develop lpr-like phenotypes in Fig. 4A and 4B, FADD control bone marrow cells readily Downloaded from akin to that of the Fas-deficient mice. However, flow cytometric differentiated into macrophages, whereas FADD2/2 bone marrow analysis revealed an unexpected phenotype. Although the initial cells showed a dramatically decreased propensity for macrophagic deletion efficiencies (5 d postinjection) in the control (45.2 6 cell production. 9.3%) and mutant mice (43.6 6 7.8%) were similar (n = 5), the To ascertain whether the defects seen in macrophage generation percentage of FADD2/2 (GFP2) cells in the bone marrow, thy- apply to other hematopoietic lineages, GFP2 FADD2/2 and 2/2 flox +/2

mus, and periphery of FADD FADD:GFP Mx1-cre mice FADD bone marrow cells were induced to differentiate into http://www.jimmunol.org/ were dramatically decreased compared with control FADD+/2 DCs by culturing with GM-CSF and IL-4 (53, 60). Following FADD:GFPﬂox Mx1-cre mice as they aged, (Fig. 3A). At 3 mo post 8 d in culture, FADD2/2 bone marrow cells generated a lower poly(I:C) injection, control mice contained more GFP2 T cells yield of bone marrow-derived DCs as determined by cell number (.50%) and GFP2 B cells (.74.7%) in the spleen and lymph when compared with FADD+/2 bone marrow (Fig. 4C,4D). by guest on September 27, 2021

FIGURE 4. In vitro bone marrow progenitor-derived macrophages and DCs. In vitro generation of macrophages from bone marrow cells isolated from FADD+/2 FADD:GFPflox Mx1-cre control and FADD2/2 FADD:GFPflox Mx1-cre mutant mice following a 7 d culture in M-CSF– containing media (A, B). DCs were generated by culturing in media containing GM-CSF and IL-4 for 8 d (C–E). Cells in the plate were photographed (A, C), cell numbers were enumerated, bar graphs generated (B, D), and flow cytometric analysis of DC populations was undertaken by staining with Abs specific for CD11b and CD11c (E). 208 FADD FUNCTION IN EARLY HEMATOPOIESIS

Additionally, a lower percentage of CD11b+CD11c+ DCs de- veloped in FADD2/2 bone marrow cultures than in FADD+/2 cultures (Fig. 4E). Taken together, these results illustrate impairment of the capability of hematopoietic progenitor cells to generate macrophages and DCs in vitro when FADD deletion is induced in the bone marrow. FADD2/2 bone marrow cells have decreased in vitro colony-forming capability Multipotent hematopoietic progenitors develop from HSCs following differentiation and loss of self-renewal potential (61). The obstruction in differentiation displayed by FADD2/2 hematopoietic progenitors (Fig. 4) may be caused by a defect in HSCs and/or the further differentiated MPPs. To investigate the differentiation capability of FADD2/2 HSCs and progenitors, in vitro CFU assays were performed. Hematopoietic progenitor cells, when cultured in a semisolid methylcellulose-based medium supplemented with suitable growth factors, proliferate and differentiate 2 2 to produce clonal clusters of maturing cells. FADD / and Downloaded from FADD+/2 bone marrow cells were cultured as a single-cell suspension within a semisolid methylcellulose matrix containing rmSCF, rmIL-3, rhIL-6, and EPO. This allowed for the suspended isolation of solitary HSCs within the matrix and the measurement of their cytokine-induced differentiation into hematopoietic pro- +/2 genitor colonies. When compared with FADD controls, mutant http://www.jimmunol.org/ FADD2/2 bone marrow cells displayed a signiﬁcantly reduced capacity to differentiate into multipotent myeloid and erythroid colonies (Fig. 5). This demonstrates that the previously observed defect in macrophage and DC development in FADD2/2 bone marrow progenitors in vitro is caused by a block in the differentiation of HSCs into further committed progenitors. The in vivo effect of bone marrow FADD deﬁciency

To determine whether the in vitro defect in bone marrow cells is by guest on September 27, 2021 applicable in vivo, we performed splenic colony-forming assays (62). FADD+/2 and FADD2/2 bone marrow cells were injected into lethally irradiated wild-type (WT) recipient C57BL/6 mice. Eight days posttransfer, mice were sacrificed, spleens were weighed and fixed, and colonies were enumerated. Gross mor- +/2 phology of the spleens in mice injected with FADD cells dif- FIGURE 5. Defects in in vitro colony formation from FADD2/2 HSCs. 2/2 fered from FADD injected mice (Fig. 6A). Spleens repopulated Bone marrow cells from FADD+/2 FADD:GFPflox Mx1-cre control and 2 2 with FADD / cells weighed less and contained fewer hemato- FADD2/2 FADD:GFPflox Mx1-cre mutant mice were cultured in a semi- 2 poietic colonies than did mice injected with FADD+/ cells (Fig. solid media containing rmSCF, rmIL-3, rhIL-6, and EPO to differentiate 6B,6C). These results show that the decreased myelopoiesis of hematopoietic stem cells for 10–14 d. Plates were photographed for FADD2/2 HSCs observed in vitro is reproducible in vivo. morphological appearance of colonies (A) and colonies scored for the In addition to myeloid differentiation, hematopoietic progeni- presence of CFU-GM, CFU-GEMM, and BFU-E (B–D). tors also develop into cells of the erythroid lineage. We have shown that FADD2/2 bone marrow progenitors are defective in in vitro generation of erythroid precursors (Fig. 5A,5D). To study whether and repopulation of lymphoid organs, GFP2 FADD+/2 and FADD functions in in vivo erythrocyte survival and development, FADD2/2 bone marrow cells were adoptively transferred into we performed hematocrit assays. Experimental mice were bled at sublethally irradiated Rag-12/2 mice to examine whether a lack of multiple time points, both prior to and following deletion, and FADD leads to defects in lymphopoiesis. Analysis of reconstituted RBC volume was measured. A significant decrease in the volume mice revealed a drastic decrease in the cellularity of lymphoid of RBCs in collected peripheral blood was seen in FADD2/2 organs in hosts reconstituted with FADD2/2 bone marrow cells FADD:GFPflox Mx1-cre mice when compared with FADD+/2 (Fig. 8A). Additionally, FADD2/2 bone marrow cells exhibited FADD:GFPflox Mx1-cre mice during the time period leading from defective reconstitution of lymphoid progenitors and immature the final injection until 6 d postinjection (Fig. 7). These results lymphocytes in the primary lymphoid organs, bone marrow, and reveal that FADD is important for the survival and development of thymus, as determined by flow cytometric analysis (Fig. 8B,8C). erythrocytes in vivo. This defect extended to the repopulation of peripheral organs such Although we demonstrated that it is required for the differen- as the spleen and peritoneal cavity with mature T and B lym- tiation of nonlymphoid cells from hematopoietic progenitors, no phocytes when compared with FADD+/2-injected counterparts distinct function has been determined for FADD with regard (Fig. 8D,8E). These results demonstrate that deletion of FADD to lymphocyte development. As transplantation of hematopoietic in bone marrow hematopoietic precursors significantly impairs progenitors into lymphopenic hosts initiates their differentiation their ability to differentiate into cells of multiple hematopoietic The Journal of Immunology 209

FIGURE 6. In vivo colony formation analyses. A, Bone marrow cells from FADD+/2 FADD: GFPflox Mx1-cre and FADD2/2 FADD:GFPflox Mx1-cre were injected into lethally irradiated B6 mice. Spleens were isolated 8 d later and fixed in Bouin’s solution. B, Weight of spleen isolated from uninjected control mice or mice injected with FADD+/2 or FADD2/2 bone marrow cells. C, Colo- nies scored from experimental mice following fixation in Bouin’s solution. Downloaded from http://www.jimmunol.org/

lineages in vivo, in addition to the previously described in vitro 2A, the absolute number of bone marrow cells was decreased in defects. FADD2/2 FADD:GFPflox Mx1-cre mutant mice when compared with FADD+/2 FADD:GFPflox Mx1-cre control mice. FADD2/2 FADD deficiency results in decreased hematopoietic progenitor progenitors displayed decreased differentiation and repopulation pools of the periphery (Fig. 3). To determine whether the deletion of 2/2 Our data thus far have shown that FADD bone marrow cells are FADD impacted the in vivo maintenance of hematopoietic pro- by guest on September 27, 2021 defective in their capability to generate multiple lineages in vitro genitors, we performed multiparameter flow cytometry analyses of or in vivo. Yet, no insight was provided as to the mechanism bone marrow cells. In FADD2/2 FADD:GFPflox Mx1-cre mutant causing this deficiency, as the defect may be due to either a flaw in mice, the HSC and MPP-enriched Lin2Sca-1+c-Kit+ (LSK) pop- the perpetuation of hematopoietic progenitor cells or an impair- ulation was significantly reduced, in comparison with FADD+/2 ment of their proliferation and differentiation. As indicated in Fig. FADD:GFPflox Mx1-cre control mice (Fig. 9A,9B). Additionally, a concomitant decrease was also detected within the Lin2c-Kit+ Sca-12 (LK) population, which contains MPP cells (Fig. 9A,9B). We further analyzed the multiple lineage progenitor populations present in the bone marrow, including the CLPs, CMPs, MEPs, and the further differentiated GMPs, to determine the effect of FADD deletion on these cells. We found that the bone marrow of FADD2/2 FADD:GFPflox Mx1-cre mice contained decreased numbers of CLP, CMP, MEP, and GMP cells (Fig. 9B). In total, these results demonstrate that FADD is required for bone marrow hematopoietic progenitor cell maintenance.

Discussion DRs are initiators of diverse signaling responses, which are involved in the regulation of homeostasis, tumor surveillance, inflammatory responses, and adaptive immune responses (5, 63). Previous studies have indicated that Fas, TNF-R1, DR3, and TRAIL-Rs (DR4 and DR5) require FADD for relaying apoptotic signals (6–8, 64–67). Nonapoptotic signals can also be induced by some of the DRs, leading to the activation of NF-kB and MAPKs. Nonetheless, DRs do not appear to play a significant role in embryos because mice 2/2 lacking each DR or mice lacking both Fas and TNF-R1 appear to FIGURE 7. Decrease in RBCs in FADD mice. Following adminis- 2/2 tration of poly(I:C), mice were retro-orbitally bled. RBC percentage in develop normally. Therefore, the embryonic defects in FADD peripheral blood from FADD+/2FADD:GFPflox Mx1-cre and FADD2/2 mice are likely due to the loss of a DR-independent function of FADD:GFPflox Mx1-cre mice was analyzed by centrifuging blood samples FADD (23, 24). Although essential in embryos, the function of and measuring hematocrit volume. *p , 0.05. FADD during hematopoiesis has remained a paradox. Initial 210 FADD FUNCTION IN EARLY HEMATOPOIESIS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 8. In vivo lymphoid reconstitution analyses. A, Cellularity of lymphoid organs isolated 8–10 wk following adoptive transfer of bone marrow cells from FADD+/2 FADD:GFPﬂox Mx1-cre and FADD2/2 FADD:GFPﬂox Mx1-cre into sublethally irradiated Rag-12/2 mice. Flow cytometric analysis of lymphocyte populations. Thymocyte populations were analyzed by CD4 and CD8 expression (B), bone marrow by B220 and IgM expression (C), spleens analyzed by immunostaining for CD3 and CD19 (D), and peritoneal cavity cells by staining with Abs for CD5 and B220 (E). n =3.*p , 0.005. analyses showed that FADD2/2 embryonic stem (ES) cell→Rag- hypothesize that FADD is required at earlier, prelineage com- 12/2 blastocyst chimeric mice contained no detectable B cells and mitment stages of hematopoiesis, with a potential function in other few T cells, suggesting a potential function for FADD in lym- hematopoietic developmental pathways as well. In this study, we phopoiesis (23). However, when postlineage commitment deletion addressed this issue by inducing the deletion of FADD in bone of FADD was initiated at the pro-B and pro/pre-T stages using the marrow cells using Mx1-cre. Our data demonstrated that FADD CD19-cre (68) and Lck-cre (69), respectively, subsequent bone plays a critical role in hematopoietic progenitors. marrow B cell development and thymic T cell development were Previously, Mx1-cre was used to induce the deletion of Mcl-1, an not affected (26, 27). The disparity between these data led us to antiapoptosis protein of the Bcl-2 family, which resulted in severe The Journal of Immunology 211

FIGURE 9. Analysis of HSCs and hematopoietic progenitors in FADD2/2 mice. A, Four-color ﬂow cytometric analysis of HSCs (Lin2Sca-1+c-Kit+) and progenitors (Lin2Sca-12c-Kit+) at day 4 after Cre induction. Results are representative of six pairs of control and mutant mice. B, Dif-

ferentiated hematopoietic progenitors were Downloaded from further analyzed by six-color ﬂow cytometry using sequential gating to study the absolute numbers of LSK, LK, CLPs (Lin2 IL-7Ra+c-KitloSca-1lo), CMPs (Lin2c-Kit+ Sca-12FcgRloCD34+), MEPs (Lin2c-Kit+ Sca-12FcgRloCD342), and GMPs (Lin2c- + 2 hi + Kit Sca-1 FcgR CD34 ) present in the http://www.jimmunol.org/ bone marrow as described (33, 34). Four to six pairs of control and mutant mice were analyzed. Horizontal bars indicate the mean values. by guest on September 27, 2021

defects in HSC survival (42). Similarly, efficient deletion of Pten depletion of the FADD2/2 population phenotype further implies in HSCs was induced using Mx1-cre, revealing a critical role a deficiency in the replenishment of various hematopoietic com- for Pten in HSC maintenance and lineage regulation (48). In our partments by FADD2/2 HSCs and progenitors. system, injection of poly(I:C) into mice induced excision of the In supporting a role for FADD in early hematopoietic de- FADD:GFPflox transgene in multiple hematopoietic organs in mice velopment stages prior to lineage commitment, we demonstrated containing no or just one endogenous FADD allele (FADD2/2 that bone marrow cells isolated from FADD2/2 mice displayed FADD:GFPflox Mx1-cre and FADD+/2 FADD:GFPflox Mx1-cre). impaired in vitro generation of macrophages and DCs (Fig. 4). Flow cytometric assessment of deletion efficiencies as well as the Furthermore, FADD2/2 bone marrow cells had diminished tracking of the GFP2 FADD2/2 mutant cells in mice were facil- ability to form colonies in vitro of various hematopoietic line- itated by using the GFP tag fused to FADD (Figs. 1–3). Deletion ages including CFU-GM, CFU-GEMM, and BFU-E (Fig. 5). occurs concurrently in both B and T cells in peripheral lymphoid These observed in vitro deficiencies were recapitulated in vivo, organs (Figs. 2, 3). Induction of FADD deletion had resulted in as FADD2/2 bone marrow cells were defective in the generation dramatic reduction of total cellularities in the bone marrow and of hematopoietic cells in vivo, following adoptive transfer into thymus (Fig. 2A). The contraction of these primary organs is likely immunodeficient hosts (Figs. 6, 8). Defects in erythroid pro- due to defects in hematopoietic progenitor cells in which FADD genitor differentiation as detected by in vitro assays may lead to deletion was induced by Mx1-cre, as such an impact was not seen reduced peripheral RBC numbers, as indicated by a deminished when FADD was deleted after lineage commitment using Lck-cre hematocrit in FADD2/2 FADD:GFPflox Mx1-cre mutant mice or CD19-cre (26, 27). Importantly, GFP2 FADD+/2 cells persist in (Fig. 7). Therefore, these data help establish a function for control FADD+/2 FADD:GFPflox Mx1-cre mice, but the GFP2 FADD in hematopoietic progenitor cells. FADD2/2 population was drastically reduced in FADD2/2 HSCs are characterized by their ability to undergo asymmetric FADD:GFPflox Mx1-cre mutant mice, particularly at 3 mo after division, with one daughter cell maintaining the characteristics of the last dose of poly(I:C) injection (Fig. 3). This time-dependent the HSC and the other committing to differentiation (70). As such, 212 FADD FUNCTION IN EARLY HEMATOPOIESIS both maintenance and differentiation of the HSC and progenitor 5. Nagata, S. 1997. Apoptosis by death factor. Cell 88: 355–365. 6. Chinnaiyan, A. M., K. O’Rourke, M. Tewari, and V. M. Dixit. 1995. FADD, populations are required for successful development and main- a novel death domain-containing protein, interacts with the death domain of Fas tenance of the hematopoietic system. A four-color flow cyto- and initiates apoptosis. Cell 81: 505–512. metric analysis of bone marrow cells revealed a dramatic re- 7. Boldin, M. P., E. E. Varfolomeev, Z. Pancer, I. L. Mett, J. H. Camonis, and D. Wallach. 1995. A novel protein that interacts with the death domain of Fas/ ductionintheHSC-enrichedLSKpopulationinFADDmutant APO1 contains a sequence motif related to the death domain. J. Biol. Chem. 270: mice (Fig. 9A,9B). In addition, FADD deletion also resulted in 7795–7798. a greatly diminished early progenitor population (LK). Further 8. Zhang, J., and A. Winoto. 1996. A mouse Fas-associated protein with homology to the human Mort1/FADD protein is essential for Fas-induced apoptosis. Mol. multicolor flow cytometric analyses demonstrated that the total Cell. Biol. 16: 2756–2763. numbers of various lineage progenitors—CLPs, CMPs, MEPs 9. Muzio, M., A. M. Chinnaiyan, F. C. Kischkel, K. O’Rourke, A. Shevchenko, and further differentiated GMPs—were also dramatically re- J. Ni, C. Scaffidi, J. D. Bretz, M. Zhang, R. Gentz, et al. 1996. FLICE, a novel 2/2 FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/ duced in FADD mutant mice (Fig. 9B). The reduction in both APO-1) death—inducing signaling complex. Cell 85: 817–827. the HSC and progenitor populations indicates a requirement for 10. Peter, M. E., and P. H. Krammer. 2003. The CD95(APO-1/Fas) DISC and be- yond. Cell Death Differ. 10: 26–35. FADD in the maintenance of early hematopoietic precursors. 11. Irmler, M., M. Thome, M. Hahne, P. Schneider, K. Hofmann, V. Steiner, FADD mediates its function during apoptotic signaling through J.-L. Bodmer M. Schro¨ter, K. Burns, C. Mattmann, et al. 1997. Inhibition of the recruitment of caspase 8 and c-FLIP to a signaling complex, death receptor signals by cellular FLIP. Nature 388: 190–195. 12. Motoyama, N., F. Wang, K. A. Roth, H. Sawa, K.-I. Nakayama, K. Nakayama, initiating a caspase cascade (6–8). FADD, caspase 8, and c-FLIP I. Negishi, S. Senju, Q. Zhang, S. Fujii, and D. Y. Loh. 1995. Massive cell death knockout mice may have embryonic heart defects (23–25, 71). of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science However, disruption of hematopoiesis may also contribute to the 267: 1506–1510. 13. Opferman, J. T., A. Letai, C. Beard, M. D. Sorcinelli, C. C. Ong, and embryonic lethality in mice lacking FADD, caspase 8, or c-FLIP. S. J. Korsmeyer. 2003. Development and maintenance of B and T lymphocytes Downloaded from 2 2 2 2 2 2 Both FADD / ES cell→Rag-1 / blastocyst and c-FLIP / requires antiapoptotic MCL-1. Nature 426: 671–676. ES cell→Rag-12/2 blastocyst chimeras displayed defective T and 14. Lindsten, T., A. J. Ross, A. King, W. X. Zong, J. C. Rathmell, H. A. Shiels, 2/2 E. Ulrich, K. G. Waymire, P. Mahar, K. Frauwirth, et al. 2000. The combined B lymphopoiesis, and caspase 8 embryos contained depleted functions of proapoptotic Bcl-2 family members bak and bax are essential for hematopoietic precursor populations, implicating these proteins as normal development of multiple tissues. Mol. Cell 6: 1389–1399. 15. Rathmell, J. C., and C. B. Thompson. 2002. Pathways of apoptosis integral regulators of hematopoiesis (23, 25, 72). Surprisingly in lymphocyte development, homeostasis, and disease. Cell 109(Suppl): S97– however, deletion of these proteins at the pro-T or pro-B cell S107. http://www.jimmunol.org/ stages of development did not inhibit the development of T and B 16. Bouillet, P., D. Metcalf, D. C. Huang, D. M. Tarlinton, T. W. Kay, F. Ko¨ntgen, J. M. Adams, and A. Strasser. 1999. Proapoptotic Bcl-2 relative Bim required for lymphoid precursors within the thymus and bone marrow, re- certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmu- spectively, bringing to light that the requisite need for these pro- nity. Science 286: 1735–1738. teins is found at earlier stages of hematopoiesis prior to lineage 17. Nagata, S., and P. Golstein. 1995. The Fas death factor. Science 267: 1449–1456. 18. Adachi, M., S. Suematsu, T. Suda, D. Watanabe, H. Fukuyama, J. Ogasawara, commitment (26–30, 73, 74). Our results detailed in this study T. Tanaka, N. Yoshida, and S. Nagata. 1996. Enhanced and accelerated lym- provide the first evidence, to our knowledge, that FADD de- phoproliferation in Fas-null mice. Proc. Natl. Acad. Sci. USA 93: 2131–2136. ficiency impairs the maintenance of hematopoietic progenitors. 19. Pfeffer, K., T. Matsuyama, T. M. Ku¨ndig, A. Wakeham, K. Kishihara, A. Shahinian, K. Wiegmann, P. S. Ohashi, M. Kro¨nke, and T. W. Mak. 1993. Interestingly, inducible deletion of caspase 8 led to defective Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to en- differentiation of hematopoietic progenitors, without affecting the dotoxic shock, yet succumb to L. monocytogenes infection. Cell 73: 457–467. by guest on September 27, 2021 maintenance of HSCs and progenitors (43). The nature of non- 20. Rothe, J., W. Lesslauer, H. Lo¨tscher, Y. Lang, P. Koebel, F. Ko¨ntgen, A. Althage, R. Zinkernagel, M. Steinmetz, and H. Bluethmann. 1993. Mice lacking the tu- apoptotic signaling pathways mediated by FADD, caspase 8, and mour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly c-FLIP has been investigated vigorously. In particular, recent susceptible to infection by Listeria monocytogenes. Nature 364: 798–802. 21. Wang, E. C. Y., A. Thern, A. Denzel, J. Kitson, S. N. Farrow, and M. J. Owen. studies have implicated a role for FADD and caspase 8 in the 2001. DR3 regulates negative selection during thymocyte development. Mol. regulation of necrosis and/or autophagy in T cells (75, 76). Fur- Cell. Biol. 21: 3451–3461. thermore, there is evidence supporting a role for these three 22. Diehl, G. E., H. H. Yue, K. Hsieh, A. A. Kuang, M. Ho, L. A. Morici, L. L. Lenz, D. Cado, L. W. Riley, and A. Winoto. 2004. TRAIL-R as a negative regulator of proteins in the regulation of the NF-kB pathways (77–79). In innate immune cell responses. Immunity 21: 877–889. summary, the DISC proteins constitute a signaling axis with di- 23. Zhang, J., D. Cado, A. Chen, N. H. Kabra, and A. Winoto. 1998. Fas-mediated verse roles mediating a variety of pathways that function in a cell- apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1. Nature 392: 296–300. specific manner during embryogenesis, hematopoiesis, and im- 24. Yeh, W.-C., J. L. Pompa, M. E. McCurrach, H.-B. Shu, A. J. Elia, A. Shahinian, mune responses. M. Ng, A. Wakeham, W. Khoo, K. Mitchell, et al. 1998. FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science 279: 1954–1958. Acknowledgments 25. Varfolomeev, E. E., M. Schuchmann, V. Luria, N. Chiannilkulchai, J. S. Beckmann, I. L. Mett, D. Rebrikov, V. M. Brodianski, O. C. Kemper, We thank Eric Ronzone and Angela Stauffer for technical help and critical O. Kollet, et al. 1998. Targeted disruption of the mouse Caspase 8 gene reading of the manuscript, Dr. Catherine Calkins for help in hematocrit anal- ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is yses, and Dr. Sean Morrison for discussion regarding enrichment/purity lethal prenatally. Immunity 9: 267–276. analyses of mouse bone marrow hematopoietic stem cells using multicolor 26. Zhang, Y., S. Rosenberg, H. Wang, H. Z. Imtiyaz, Y. J. Hou, and J. Zhang. 2005. Conditional Fas-associated death domain protein (FADD): GFP knockout mice flow cytometry. reveal FADD is dispensable in thymic development but essential in peripheral T cell homeostasis. J. Immunol. 175: 3033–3044. 27. Imtiyaz, H. Z., S. Rosenberg, Y. Zhang, Z. S. Rahman, Y. J. Hou, T. Manser, and Disclosures J. Zhang. 2006. The Fas-associated death domain protein is required in apoptosis The authors have no financial conflicts of interest. and TLR-induced proliferative responses in B cells. J. Immunol. 176: 6852– 6861. 28. Salmena, L., B. Lemmers, A. Hakem, E. Matysiak-Zablocki, K. Murakami, References P. Y. Au, D. M. Berry, L. Tamblyn, A. Shehabeldin, E. Migon, et al. 2003. Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. Nature 1. Meier, P., A. Finch, and G. Evan. 2000. Apoptosis in development. 407: Genes Dev. 17: 883–895. 796–801. 29. Beisner, D. R., I. L. Ch’en, R. V. Kolla, A. Hoffmann, and S. M. Hedrick. 2005. 2. Opferman, J. T., and S. J. Korsmeyer. 2003. Apoptosis in the development and Cutting edge: innate immunity conferred by B cells is regulated by caspase-8. J. maintenance of the immune system. Nat. Immunol. 4: 410–415. Immunol. 175: 3469–3473. 3. Sprick, M. R., and H. Walczak. 2004. The interplay between the Bcl-2 family 30. Lemmers, B., L. Salmena, N. Bide`re, H. Su, E. Matysiak-Zablocki, and death receptor-mediated apoptosis. Biochim. Biophys. Acta 1644: 125–132. K. Murakami, P. S. Ohashi, A. Jurisicova, M. Lenardo, R. Hakem, and 4. Strasser, A., P. J. Jost, and S. Nagata. 2009. The many roles of FAS receptor A. Hakem. 2007. Essential role for caspase-8 in Toll-like receptors and signaling in the immune system. Immunity 30: 180–192. NFkappaB signaling. J. Biol. Chem. 282: 7416–7423. The Journal of Immunology 213

31. Osawa, M., K. Hanada, H. Hamada, and H. Nakauchi. 1996. Long-term lym- kinase IV regulates hematopoietic stem cell maintenance. J. Biol. Chem. 280: phohematopoietic reconstitution by a single CD34-low/negative hematopoietic 33101–33108. stem cell. Science 273: 242–245. 56. Siminovitch, L., E. A. McCulloch, and J. E. Till. 1963. The Distribution of 32. Nilsson, S. K., H. M. Johnston, and J. A. Coverdale. 2001. Spatial localization of Colony-Forming Cells among Spleen Colonies. J. Cell. Physiol. 62: 327–336. transplanted hemopoietic stem cells: inferences for the localization of stem cell 57. Caulfield, M. J., D. Stanko, and C. Calkins. 1989. Characterization of the niches. Blood 97: 2293–2299. spontaneous autoimmune (anti-erythrocyte) response in NZB mice using 33. Kondo, M., I. L. Weissman, and K. Akashi. 1997. Identification of clonogenic a pathogenic monoclonal autoantibody and its anti-idiotype. Immunology 66: common lymphoid progenitors in mouse bone marrow. Cell 91: 661–672. 233–237. 34. Akashi, K., D. Traver, T. Miyamoto, and I. L. Weissman. 2000. A clonogenic 58. Stranges, P. B., J. Watson, C. J. Cooper, C. M. Choisy-Rossi, A. C. Stonebraker, common myeloid progenitor that gives rise to all myeloid lineages. Nature 404: R. A. Beighton, H. Hartig, J. P. Sundberg, S. Servick, G. Kaufmann, et al. 2007. 193–197. Elimination of antigen-presenting cells and autoreactive T cells by Fas con- 35. Pelayo, R., R. Welner, S. S. Perry, J. Huang, Y. Baba, T. Yokota, and tributes to prevention of autoimmunity. Immunity 26: 629–641. P. W. Kincade. 2005. Lymphoid progenitors and primary routes to 59. Stanley, E. R. 1985. The macrophage colony-stimulating factor, CSF-1. Methods becoming cells of the immune system. Curr. Opin. Immunol. 17: 100–107. Enzymol. 116: 564–587. 36. Laslo, P., J. M. Pongubala, D. W. Lancki, and H. Singh. 2008. Gene regulatory 60. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, networks directing myeloid and lymphoid cell fates within the immune system. and R. M. Steinman. 1992. Generation of large numbers of dendritic cells from Semin. Immunol. 20: 228–235. mouse bone marrow cultures supplemented with granulocyte/macrophage 37. Zlotoff, D. A., B. A. Schwarz, and A. Bhandoola. 2008. The long road to the colony-stimulating factor. J. Exp. Med. 176: 1693–1702. thymus: the generation, mobilization, and circulation of T-cell progenitors in 61. Orkin, S. H. 2000. Diversification of haematopoietic stem cells to specific lin- mouse and man. Semin. Immunopathol. 30: 371–382. eages. Nat. Rev. Genet. 1: 57–64. 38. Laiosa, C. V., M. Stadtfeld, and T. Graf. 2006. Determinants of lymphoid- 62. Till, J. E., and E. A. McCulloch. 1961. A direct measurement of the radiation myeloid lineage diversification. Annu. Rev. Immunol. 24: 705–738. sensitivity of normal mouse bone marrow cells. Radiat. Res. 14: 213–222. 39. Inaba, K., M. Inaba, M. Deguchi, K. Hagi, R. Yasumizu, S. Ikehara, 63. Ashkenazi, A., and V. M. Dixit. 1998. Death receptors: signaling and modula- S. Muramatsu, and R. M. Steinman. 1993. Granulocytes, macrophages, and tion. Science 281: 1305–1308. dendritic cells arise from a common major histocompatibility complex class II- 64. Hsu, H., H.-B. Shu, M.-G. Pan, and D. V. Goeddel. 1996. TRADD-TRAF2 and negative progenitor in mouse bone marrow. Proc. Natl. Acad. Sci. USA 90: TRADD-FADD interactions define two distinct TNF receptor 1 signal trans- Downloaded from 3038–3042. duction pathways. Cell 84: 299–308. 40. Ma, A., J. C. Pena, B. Chang, E. Margosian, L. Davidson, F. W. Alt, and 65. Chinnaiyan, A. M., K. O’Rourke, G.-L. Yu, R. H. Lyons, M. Garg, D. R. Duan, C. B. Thompson. 1995. Bclx regulates the survival of double-positive thymo- L. Xing, R. Gentz, J. Ni, and V. M. Dixit. 1996. Signal transduction by DR3, cytes. Proc. Natl. Acad. Sci. USA 92: 4763–4767. a death domain-containing receptor related to TNFR-1 and CD95. Science 274: 41. Motoyama, N., T. Kimura, T. Takahashi, T. Watanabe, and T. Nakano. 1999. bcl- 990–992. x prevents apoptotic cell death of both primitive and definitive erythrocytes at the 66. Kuang, A. A., G. E. Diehl, J. Zhang, and A. Winoto. 2000. FADD is required for end of maturation. J. Exp. Med. 189: 1691–1698. DR4- and DR5-mediated apoptosis: lack of trail-induced apoptosis in FADD-

42. Opferman, J. T., H. Iwasaki, C. C. Ong, H. Suh, S. Mizuno, K. Akashi, and deficient mouse embryonic fibroblasts. J. Biol. Chem. 275: 25065–25068. http://www.jimmunol.org/ S. J. Korsmeyer. 2005. Obligate role of anti-apoptotic MCL-1 in the survival of 67. Kischkel, F. C., D. A. Lawrence, A. Chuntharapai, P. Schow, K. J. Kim, and hematopoietic stem cells. Science 307: 1101–1104. A. Ashkenazi. 2000. Apo2L/TRAIL-dependent recruitment of endogenous 43. Kang, T. B., T. Ben-Moshe, E. E. Varfolomeev, Y. Pewzner-Jung, N. Yogev, FADD and caspase-8 to death receptors 4 and 5. Immunity 12: 611–620. A. Jurewicz, A. Waisman, O. Brenner, R. Haffner, E. Gustafsson, et al. 2004. 68. Rickert, R. C., J. Roes, and K. Rajewsky. 1997. B lymphocyte-specific, Cre- Caspase-8 serves both apoptotic and nonapoptotic roles. J. Immunol. 173: 2976– mediated mutagenesis in mice. Nucleic Acids Res. 25: 1317–1318. 2984. 69. Lee, P. P., D. R. Fitzpatrick, C. Beard, H. K. Jessup, S. Lehar, K. W. Makar, 44. Pellegrini, M., S. Bath, V. S. Marsden, D. C. Huang, D. Metcalf, A. W. Harris, M. Pe´rez-Melgosa, M. T. Sweetser, M. S. Schlissel, S. Nguyen, et al. 2001. A and A. Strasser. 2005. FADD and caspase-8 are required for cytokine-induced critical role for Dnmt1 and DNA methylation in T cell development, function, proliferation of hemopoietic progenitor cells. Blood 106: 1581–1589. and survival. Immunity 15: 763–774. 45. Ku¨hn, R., F. Schwenk, M. Aguet, and K. Rajewsky. 1995. Inducible gene tar- 70. Domen, J., and I. L. Weissman. 1999. Self-renewal, differentiation or death: geting in mice. Science 269: 1427–1429. regulation and manipulation of hematopoietic stem cell fate. Mol. Med. Today 5:

46. Hock, H., E. Meade, S. Medeiros, J. W. Schindler, P. J. Valk, Y. Fujiwara, and 201–208. by guest on September 27, 2021 S. H. Orkin. 2004. Tel/Etv6 is an essential and selective regulator of adult he- 71. Yeh, W.-C., A. Itie, A. J. Elia, M. Ng, H.-B. Shu, A. Wakeham, C. Mirtsos, matopoietic stem cell survival. Genes Dev. 18: 2336–2341. N. Suzuki, M. Bonnard, D. V. Goeddel, and T. W. Mak. 2000. Requirement for 47. Yilmaz, O. H., R. Valdez, B. K. Theisen, W. Guo, D. O. Ferguson, H. Wu, and Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embry- S. J. Morrison. 2006. Pten dependence distinguishes haematopoietic stem cells onic development. Immunity 12: 633–642. from leukaemia-initiating cells. Nature 441: 475–482. 72. Chau, H., V. Wong, N. J. Chen, H. L. Huang, W. J. Lin, C. Mirtsos, A. R. Elford, 48. Zhang, J., J. C. Grindley, T. Yin, S. Jayasinghe, X. C. He, J. T. Ross, J. S. Haug, M. Bonnard, A. Wakeham, A. I. You-Ten, et al. 2005. Cellular FLICE-inhibitory D. Rupp, K. S. Porter-Westpfahl, L. M. Wiedemann, et al. 2006. PTEN main- protein is required for T cell survival and cycling. J. Exp. Med. 202: 405–413. tains haematopoietic stem cells and acts in lineage choice and leukaemia pre- 73. Zhang, N., and Y. W. He. 2005. An essential role for c-FLIP in the efﬁcient vention. Nature 441: 518–522. development of mature T lymphocytes. J. Exp. Med. 202: 395–404. 49. Austin, P. E., E. A. McCulloch, and J. E. Till. 1971. Characterization of the 74. Zhang, H., S. Rosenberg, F. J. Coffey, Y. W. He, T. Manser, R. R. Hardy, and factor in L-cell conditioned medium capable of stimulating colony formation by J. Zhang. 2009. A role for cFLIP in B cell proliferation and stress MAPK reg- mouse marrow cells in culture. J. Cell. Physiol. 77: 121–134. ulation. J. Immunol. 182: 207–215. 50. Zhang, X., R. Goncalves, and D. M. Mosser. 2008. The isolation and charac- 75. Ch’en, I. L., D. R. Beisner, A. Degterev, C. Lynch, J. Yuan, A. Hoffmann, and terization of murine macrophages. Curr. Protoc. Immunol. Chapter 14: Unit 14.1. S. M. Hedrick. 2008. Antigen-mediated T cell expansion regulated by parallel 51. Higashi, N., A. Morikawa, K. Fujioka, Y. Fujita, Y. Sano, M. Miyata-Takeuchi, pathways of death. Proc. Natl. Acad. Sci. USA 105: 17463–17468. N. Suzuki, and T. Irimura. 2002. Human macrophage lectin speciﬁc for galactose/ 76. Bell, B. D., S. Leverrier, B. M. Weist, R. H. Newton, A. F. Arechiga, N-acetylgalactosamine is a marker for cells at an intermediate stage in their dif- K. A. Luhrs, N. S. Morrissette, and C. M. Walsh. 2008. FADD and caspase-8 ferentiation from monocytes into macrophages. Int. Immunol. 14: 545–554. control the outcome of autophagic signaling in proliferating T cells. Proc. Natl. 52. Schreurs, M. W., A. A. Eggert, A. J. de Boer, C. G. Figdor, and G. J. Adema. Acad. Sci. USA 105: 16677–16682. 1999. Generation and functional characterization of mouse monocyte-derived 77. Su, H., N. Bide`re, L. Zheng, A. Cubre, K. Sakai, J. Dale, L. Salmena, R. Hakem, dendritic cells. Eur. J. Immunol. 29: 2835–2841. S. Straus, and M. Lenardo. 2005. Requirement for caspase-8 in NF-kappaB 53. Inaba, K., W. J. Swiggard, R. M. Steinman, N. Romani, G. Schuler, and C. Brinster. activation by antigen receptor. Science 307: 1465–1468. 2009. Isolation of dendritic cells. Curr. Protoc. Immunol. Chapter 3: Unit 3.7. 78. Misra, R. S., J. Q. Russell, A. Koenig, J. A. Hinshaw-Makepeace, R. Wen, 54. Ito, C. Y., C. Y. Li, A. Bernstein, J. E. Dick, and W. L. Stanford. 2003. He- D. Wang, H. Huo, D. R. Littman, U. Ferch, J. Ruland, et al. 2007. Caspase-8 and matopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice. Blood c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activa- 101: 517–523. tion. J. Biol. Chem. 282: 19365–19374. 55. Kitsos, C. M., U. Sankar, M. Illario, J. M. Colomer-Font, A. W. Duncan, 79. Hu, W. H., H. Johnson, and H. B. Shu. 2000. Activation of NF-kappaB by T. J. Ribar, T. Reya, and A. R. Means. 2005. Calmodulin-dependent protein FADD, Casper, and caspase-8. J. Biol. Chem. 275: 10838–10844.